Scaling of fibre area and fibre glycogen concentration in the hindlimb musculature of monitor lizards: implications for locomotor performance with increasing body size.

A considerable biomechanical challenge faces larger terrestrial animals as the demands of body support scale with body mass (Mb), while muscle force capacity is proportional to muscle cross-sectional area, which scales with Mb2/3. How muscles adjust to this challenge might be best understood by examining varanids, which vary by five orders of magnitude in size without substantial changes in posture or body proportions. Muscle mass, fascicle length and physiological cross-sectional area all scale with positive allometry, but it remains unclear, however, how muscles become larger in this clade. Do larger varanids have more muscle fibres, or does individual fibre cross-sectional area (fCSA) increase? It is also unknown if larger animals compensate by increasing the proportion of fast-twitch (higher glycogen concentration) fibres, which can produce higher force per unit area than slow-twitch fibres. We investigated muscle fibre area and glycogen concentration in hindlimb muscles from varanids ranging from 105 g to 40,000 g. We found that fCSA increased with modest positive scaling against body mass (Mb0.197) among all our samples, and ∝Mb0.278 among a subset of our data consisting of never-frozen samples only. The proportion of low-glycogen fibres decreased significantly in some muscles but not others. We compared our results with the scaling of fCSA in different groups. Considering species means, fCSA scaled more steeply in invertebrates (∝Mb0.575), fish (∝Mb0.347) and other reptiles (∝Mb0.308) compared with varanids (∝Mb0.267), which had a slightly higher scaling exponent than birds (∝Mb0.134) and mammals (∝Mb0.122). This suggests that, while fCSA generally increases with body size, the extent of this scaling is taxon specific, and may relate to broad differences in locomotor function, metabolism and habitat between different clades.

Modular lung ventilation in Boa constrictor.

The evolution of constriction and of large prey ingestion within snakes are key innovations that may explain the remarkable diversity, distribution and ecological scope of this clade, relative to other elongate vertebrates. However, these behaviors may have simultaneously hindered lung ventilation such that early snakes may have had to circumvent these mechanical constraints before those behaviors could evolve. Here, we demonstrate that Boa constrictor can modulate which specific segments of ribs are used to ventilate the lung in response to physically hindered body wall motions. We show that the modular actuation of specific segments of ribs likely results from active recruitment or quiescence of derived accessory musculature. We hypothesize that constriction and large prey ingestion were unlikely to have evolved without modular lung ventilation because of their interference with lung ventilation, high metabolic demands and reliance on sustained lung convection. This study provides a new perspective on snake evolution and suggests that modular lung ventilation evolved during or prior to constriction and large prey ingestion, facilitating snakes' remarkable radiation relative to other elongate vertebrates.

The scaling of ground reaction forces and duty factor in monitor lizards: implications for locomotion in sprawling tetrapods.

Geometric scaling predicts a major challenge for legged, terrestrial locomotion. Locomotor support requirements scale identically with body mass (α M1), while force-generation capacity should scale α M2/3 as it depends on muscle cross-sectional area. Mammals compensate with more upright limb postures at larger sizes, but it remains unknown how sprawling tetrapods deal with this challenge. Varanid lizards are an ideal group to address this question because they cover an enormous body size range while maintaining a similar bent-limb posture and body proportions. This study reports the scaling of ground reaction forces and duty factor for varanid lizards ranging from 7 g to 37 kg. Impulses (force×time) (α M0.99-1.34) and peak forces (α M0.73-1.00) scaled higher than expected. Duty factor scaled α M0.04 and was higher for the hindlimb than the forelimb. The proportion of vertical impulse to total impulse increased with body size, and impulses decreased while peak forces increased with speed.

Monitoring muscle over three orders of magnitude: Widespread positive allometry among locomotor and body support musculature in the pectoral girdle of varanid lizards (Varanidae).

There is a functional trade-off in the design of skeletal muscle. Muscle strength depends on the number of muscle fibers in parallel, while shortening velocity and operational distance depend on fascicle length, leading to a trade-off between the maximum force a muscle can produce and its ability to change length and contract rapidly. This trade-off becomes even more pronounced as animals increase in size because muscle strength scales with area (length2 ) while body mass scales with volume (length3 ). In order to understand this muscle trade-off and how animals deal with the biomechanical consequences of size, we investigated muscle properties in the pectoral girdle of varanid lizards. Varanids are an ideal group to study the scaling of muscle properties because they retain similar body proportions and posture across five orders of magnitude in body mass and are highly active, terrestrially adapted reptiles. We measured muscle mass, physiological cross-sectional area, fascicle length, proximal and distal tendon lengths, and proximal and distal moment arms for 27 pectoral girdle muscles in 13 individuals across 8 species ranging from 64 g to 40 kg. Standard and phylogenetically informed reduced major axis regression was used to investigate how muscle architecture properties scale with body size. Allometric growth was widespread for muscle mass (scaling exponent >1), physiological cross-sectional area (scaling exponent >0.66), but not tendon length (scaling exponent >0.33). Positive allometry for muscle mass was universal among muscles responsible for translating the trunk forward and flexing the elbow, and nearly universal among humeral protractors and wrist flexors. Positive allometry for PCSA was also common among trunk translators and humeral protractors, though less so than muscle mass. Positive scaling for fascicle length was not widespread, but common among humeral protractors. A higher proportion of pectoral girdle muscles scaled with positive allometry than our previous work showed for the pelvic girdle, suggesting that the center of mass may move cranially with body size in varanids, or that the pectoral girdle may assume a more dominant role in locomotion in larger species. Scaling exponents for physiological cross-sectional area among muscles primarily associated with propulsion or with a dual role were generally higher than those associated primarily with support against gravity, suggesting that locomotor demands have at least an equal influence on muscle architecture as body support. Overall, these results suggest that larger varanids compensate for the increased biomechanical demands of locomotion and body support at higher body sizes by developing larger pectoral muscles with higher physiological cross-sectional areas. The isometric scaling rates for fascicle length among locomotion-oriented pectoral girdle muscles suggest that larger varanids may be forced to use shorter stride lengths, but this problem may be circumvented by increases in limb excursion afforded by the sliding coracosternal joint.

Unidirectional pulmonary airflow patterns in the savannah monitor lizard.

The unidirectional airflow patterns in the lungs of birds have long been considered a unique and specialized trait associated with the oxygen demands of flying, their endothermic metabolism and unusual pulmonary architecture. However, the discovery of similar flow patterns in the lungs of crocodilians indicates that this character is probably ancestral for all archosaurs--the group that includes extant birds and crocodilians as well as their extinct relatives, such as pterosaurs and dinosaurs. Unidirectional flow in birds results from aerodynamic valves, rather than from sphincters or other physical mechanisms, and similar aerodynamic valves seem to be present in crocodilians. The anatomical and developmental similarities in the primary and secondary bronchi of birds and crocodilians suggest that these structures and airflow patterns may be homologous. The origin of this pattern is at least as old as the split between crocodilians and birds, which occurred in the Triassic period. Alternatively, this pattern of flow may be even older; this hypothesis can be tested by investigating patterns of airflow in members of the outgroup to birds and crocodilians, the Lepidosauromorpha (tuatara, lizards and snakes). Here we demonstrate region-specific unidirectional airflow in the lungs of the savannah monitor lizard (Varanus exanthematicus). The presence of unidirectional flow in the lungs of V. exanthematicus thus gives rise to two possible evolutionary scenarios: either unidirectional airflow evolved independently in archosaurs and monitor lizards, or these flow patterns are homologous in archosaurs and V. exanthematicus, having evolved only once in ancestral diapsids (the clade encompassing snakes, lizards, crocodilians and birds). If unidirectional airflow is plesiomorphic for Diapsida, this respiratory character can be reconstructed for extinct diapsids, and evolved in a small ectothermic tetrapod during the Palaeozoic era at least a hundred million years before the origin of birds.

The axial anatomy of monitor lizards (Varanidae).

Because the musculoskeletal anatomy of the trunk is the framework for the behaviors of locomotion, ventilation, and body support in lepidosaurs, comparative study of trunk anatomy in this group is critical for unraveling the selective pressures leading to extant diversity in axial form and function among vertebrates. This work uses gross dissection and computed tomography to describe the muscular and skeletal anatomy of the trunk of varanid lizards (Varanidae, Anguimorpha). Gross muscle dissections were conducted to investigate the axial muscular anatomy of Varanus exanthematicus, Varanus giganteus, Varanus rosenbergi, and Varanus panoptes. Computed tomography scans of these and additional varanid lizards from the Varanus and Odatria subgenera were conducted to investigate rib and vertebral number and gross morphology. The number of vertebrae differs between species, with 27-35 presacral and 47-137 postsacral vertebrae. Although the number of floating and abdominal ribs in varanids is variable, most species examined have three to four cervical ribs and three true ribs. Attachment and insertion points of the epaxial and hypaxial musculature are detailed. The body wall has four main hypaxial layers, from superficial to deep: oliquus externus, intercostalis externi, intercostalis internii, and transversus. Varanids differ from other investigated lepidosaurs in having supracostalis dorsus brevis (epaxial) and levator costae (hypaxial), which independently connect each rib to the vertebral column. Although more basic muscle descriptions of the body wall in reptiles are needed, comparisons with the condition in the green iguana (Iguana iguana) can be made.

Breathing with floating ribs: XROMM analysis of lung ventilation in savannah monitor lizards.

The structures and functions of the vertebrate lung and trunk are linked through the act of ventilation, but the connections between these structures and functions are poorly understood. We used X-ray reconstruction of moving morphology (XROMM) to measure rib kinematics during lung ventilation in three savannah monitor lizards (Varanus exanthematicus). All of the dorsal ribs, including the floating ribs, contributed to ventilation; the magnitude and kinematic pattern showed no detectable cranial-to-caudal gradient. The true ribs acted as two rigid bodies connected by flexible cartilage, with the vertebral rib and ventromedial shaft of each sternal rib remaining rigid and the cartilage between them forming a flexible intracostal joint. Rib rotations can be decomposed into bucket handle rotation around a dorsoventral axis, pump handle rotation around a mediolateral axis and caliper motion around a craniocaudal axis. Dorsal rib motion was dominated by roughly equal contributions of bucket and pump rotation in two individuals and by bucket rotation in the third individual. The recruitment of floating ribs during ventilation in monitor lizards is strikingly different from the situation in iguanas, where only the first few true ribs contribute to breathing. This difference may be related to the design of the pulmonary system and life history traits in these two species. Motion of the floating ribs may maximize ventilation of the caudally and ventrolaterally positioned compliant saccular chambers in the lungs of varanids, while restriction of ventilation to a few true ribs may maximize crypsis in iguanas.

New insight into the evolution of the vertebrate respiratory system and the discovery of unidirectional airflow in iguana lungs.

The generally accepted framework for the evolution of a key feature of the avian respiratory system, unidirectional airflow, is that it is an adaptation for efficiency of gas exchange and expanded aerobic capacities, and therefore it has historically been viewed as important to the ability of birds to fly and to maintain an endothermic metabolism. This pattern of flow has been presumed to arise from specific features of the respiratory system, such as an enclosed intrapulmonary bronchus and parabronchi. Here we show unidirectional airflow in the green iguana, a lizard with a strikingly different natural history from that of birds and lacking these anatomical features. This discovery indicates a paradigm shift is needed. The selective drivers of the trait, its date of origin, and the fundamental aerodynamic mechanisms by which unidirectional flow arises must be reassessed to be congruent with the natural history of this lineage. Unidirectional flow may serve functions other than expanded aerobic capacity; it may have been present in the ancestral diapsid; and it can occur in structurally simple lungs.

Tail Base Deflection but not Tail Curvature Varies with Speed in Lizards: Results from an Automated Tracking Analysis Pipeline.

Tail movement is an important component of vertebrate locomotion and likely contributes to dynamic stability during steady-state locomotion. Previous results suggest that the tail plays a significant role in lizard locomotion, but little data are available on tail motion during locomotion and how it differs with morphological, ecological, and phylogenetic parameters. We collected high-speed vertical climbing and horizontal locomotion video data from 43 lizard species from four taxonomic groups (Agamidae, Gekkota, Scincidae, and Varanidae) across four habitats. We introduce a new semi-automated and generalizable analysis pipeline for tail and spine motion analysis including markerless pose-estimation, semi-automated kinematic recognition, and muti-species data analysis. We found that step length relative to snout-vent length (SVL) increased with tail length relative to SVL. Examining spine cycles agnostic to limb stride phase, we found that ranges of inter-tail bending compared with inter-spine bending increased with relative tail length, while ranges of tail deflection relative to spine deflection increased with relative speed. Considering stepwise strides, we found the angular velocity and acceleration of the tail center of mass increased with relative speed. These results will provide general insights into the biomechanics of tails in sprawling locomotion enabling biomimetic applications in robotics, and a better understanding of vertebrate form and function. We look forward to adding more species, behaviors, and locomotor speeds to our analysis pipeline through collaboration with other research groups.

Virtual and augmented reality: New tools for visualizing, analyzing, and communicating complex morphology.

Virtual and augmented reality (VR/AR) are new technologies with the power to revolutionize the study of morphology. Modern imaging approaches such as computed tomography, laser scanning, and photogrammetry have opened up a new digital world, enabling researchers to share and analyze morphological data electronically and in great detail. Because this digital data exists on a computer screen, however, it can remain difficult to understand and unintuitive to interact with. VR/AR technologies bridge the analog-to-digital divide by presenting 3D data to users in a very similar way to how they would interact with actual anatomy, while also providing a more immersive experience and greater possibilities for exploration. This manuscript describes VR/AR hardware, software, and techniques, and is designed to give practicing morphologists and educators a primer on using these technologies in their research, pedagogy, and communication to a wide variety of audiences. We also include a series of case studies from the presentations and workshop given at the 2019 International Congress of Vertebrate Morphology, and suggest best practices for the use of VR/AR in comparative morphology.

Computational Fluid Dynamics Reveals a Unique Net Unidirectional Pattern of Pulmonary Airflow in the Savannah Monitor Lizard (Varanus exanthematicus).

This report models pulmonary airflow in the savannah monitor (Varanus exanthematicus) using computational fluid dynamics simulations, which are based on computed tomography data. Simulations were validated by visualizing the flow of aerosolized lipids in excised lungs with good but not perfect agreement. The lung of this lizard has numerous successive bronchi branching off a long intrapulmonary bronchus, which are interconnected by intercameral perforations. Unidirectional flow has been documented in the lateral secondary bronchi of the savannah monitor, but patterns of airflow in the rest of the lung remain unknown, hindering our understanding of the evolution of pulmonary patterns of airflow in tetrapods. These results indicate that the lung contains a unique net unidirectional flow, where the overall flow scheme is similar during expiration and late inspiration, but dissimilar during early inspiration. Air is transported net caudally through the intrapulmonary bronchus and net craniad through secondary bronchi, much like the pattern of flow in birds. The simulations show that many chambers feature flow in multiple directions during parts of the respiratory cycle, but some regions also show robust unidirectional airflow. Air moves craniad through secondary bronchi and between adjacent secondary bronchi through intercameral perforations. The first secondary bronchus, the hilar-cranial bronchus, contains tidal flow that may improve ventilation of the central and dorsal lung parenchyma. These results expand our understanding of flow patterns in varanid lungs and suggest lungs with net unidirectional flow as an evolutionary pathway between tidal flow and complete unidirectional flow in multicameral lungs. Anat Rec, 2020. © 2019 American Association for Anatomy Anat Rec, 303:1768-1791, 2020. © 2019 American Association for Anatomy.

Locomotor rib kinematics in two species of lizards and a new hypothesis for the evolution of aspiration breathing in amniotes.

Most lizards walk and run with a sprawling gait in which the limbs are partly advanced by lateral undulation of the axial skeleton. Ribs and vertebrae are integral to this locomotor mode, but 3D motion of the axial skeleton has not been reported for lizard locomotion. Here, we use XROMM to quantify the relative motions of the vertebrae and ribs during slow treadmill locomotion in three savannah monitor lizards (Varanus exanthematicus) and three Argentine black and white tegus (Salvator merianae). To isolate locomotion, we selected strides with no concurrent lung ventilation. Rib rotations can be decomposed into bucket-handle rotation around a dorsoventral axis, pump-handle rotation around a mediolateral axis, and caliper rotations around a craniocaudal axis. During locomotion, every rib measured in both species rotated substantially around its costovertebral joint (8-17 degrees, summed across bucket, pump and caliper rotations). In all individuals from both species, the middle ribs rotated cranially through bucket and pump-handle motion during the propulsive phase of the ipsilateral forelimb. Axial kinematics during swing phase of the ipsilateral forelimb were mirror images of the propulsive phase. Although further work is needed to establish what causes these rib motions, active contraction of the hypaxial musculature may be at least partly responsible. Unilateral locomotor rib movements are remarkably similar to the bilateral pattern used for lung ventilation, suggesting a new hypothesis that rib motion during locomotion may have been an exaptation for the evolution of costal aspiration breathing in stem amniotes.

Osteology of the Late Triassic Bipedal Archosaur Poposaurus gracilis (Archosauria: Pseudosuchia) from Western North America.

Poposaurus gracilis is a bipedal pseudosuchian archosaur that has been poorly understood since the discovery of the holotype fragmentary partial postcranial skeleton in 1915. Poposaurus. gracilis is a member of Poposauroidea, an unusually morphologically divergent clade of pseudosuchians containing taxa that are bipedal, quadrupedal, toothed, edentulous, and some individuals with elongated thoracic neural spines (i.e., sails). In 2003, a well preserved, fully articulated, and nearly complete postcranial skeleton of P. gracilis was discovered with some fragmentary cranial elements from the Upper Triassic Chinle Formation of Grand Staircase-Escalante National Monument of southern Utah, USA. The aim of this work is to describe the osteology of this specimen in detail and compare P. gracilis to other closely related pseudosuchian archosaurs. The open neurocentral sutures throughout the majority of the vertebral column, the small size of this individual, and the presence of seven evenly spaced cyclic growth marks in the histologically sectioned femur indicate that this specimen was a skeletally immature juvenile, or subadult when it died. The pes of P. gracilis contains multiple skeletal adaptations and osteological correlates for soft tissue structures that support a hypothesis of digitigrady for this taxon. When coupled with the numerous postcranial characters associated with cursoriality, and the many anatomical traits convergent with theropod dinosaurs, this animal likely occupied a similar ecological niche with contemporaneous theropods during the Late Triassic Period. Anat Rec, 303:874-917, 2020. © 2019 American Association for Anatomy.

Pulmonary Smooth Muscle in Vertebrates: A Comparative Review of Structure and Function.

Although the airways of vertebrates are diverse in shape, complexity, and function, they all contain visceral smooth muscle. The morphology, function, and innervation of this tissue in airways is reviewed in actinopterygians, lungfish, amphibians, non-avian reptiles, birds, and mammals. Smooth muscle was likely involved in tension regulation ancestrally, and may serve to assist lung emptying in fishes and aquatic amphibians, as well as maintain internal lung structure. In certain non-avian reptiles and anurans antagonistic smooth muscle fibers may contribute to intrapulmonary gas mixing. In mammals and birds, smooth muscle regulates airway caliber, and may be important in controlling the distribution of ventilation at rest and exercise, or during thermoregulatory and vocal hyperventilation. Airway smooth muscle is controlled by the autonomic nervous system: cranial cholinergic innervation generally causes excitation, cranial non-adrenergic, non-cholinergic innervation causes inhibition, and spinal adrenergic (SA) input causes species-specific, often heterogeneous contractions and relaxations.

Unidirectional pulmonary airflow in vertebrates: a review of structure, function, and evolution.

Mechanisms explaining unidirectional pulmonary airflow in birds, a condition where lung gases flow in a consistent direction during both inspiration and expiration in some parts of the lung, were suggested as early as the first part of the twentieth century and unidirectional pulmonary airflow has been discovered recently in crocodilians and squamates. Our knowledge of the functional anatomy, fluid dynamics, and significance of this trait is reviewed. The preponderance of the data indicates that unidirectional airflow is maintained by means of convective inertia in inspiratory and expiratory aerodynamic valves in birds. The study of flow patterns in non-avian reptiles is just beginning, but inspiratory aerodynamic valving likely also plays an important role in controlling flow direction in these lungs. Although highly efficient counter and cross-current blood-gas exchange arrangements are possible in lungs with unidirectional airflow, very few experiments have investigated blood-gas exchange mechanisms in the bird lung and blood-gas arrangements in the lungs of non-avian reptiles are completely unknown. The presence of unidirectional airflow in non-volant ectotherms voids the traditional hypothesis that this trait evolved to supply the high aerobic demands of flight and endothermy, and there is a need for new scenarios in our understanding of lung evolution. The potential value of unidirectional pulmonary airflow for allowing economic lung gas mixing, facilitating lung gas washout, and providing for adequate gas exchange during hypoxic conditions is discussed.